EP1265986B1 - Procede permettant de produire i in vitro /i du tissu cartilagineux ou osseux tridimensionnel vivant - Google Patents

Procede permettant de produire i in vitro /i du tissu cartilagineux ou osseux tridimensionnel vivant Download PDF

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Publication number
EP1265986B1
EP1265986B1 EP01913858.5A EP01913858A EP1265986B1 EP 1265986 B1 EP1265986 B1 EP 1265986B1 EP 01913858 A EP01913858 A EP 01913858A EP 1265986 B1 EP1265986 B1 EP 1265986B1
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Prior art keywords
cells
tissue
cartilage
cell
bone
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Expired - Lifetime
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EP01913858.5A
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German (de)
English (en)
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EP1265986A2 (fr
Inventor
Jeanette Libera
Ursula Anderer
Karl-Gerd Fritsch
Olivera Josimovic-Alasevic
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Co Don AG
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Co Don AG
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Priority to CY20161100246T priority Critical patent/CY1117464T1/el
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the invention relates to a very simple process for the in vitro production of a cell aggregate of three-dimensional, vital and mechanically stable cartilage or bone tissue and its use as a transplantation material for the treatment of cartilage or bone defects and of degenerative diseases, e.g. Rheumatism or arthrosis and its use for the testing of active ingredients and physical factors.
  • a variety of cartilage and bone tissues can be produced, e.g. hyaline, elastic, fibrous or connective tissue cartilages such as articular cartilage, nasal cartilage, meniscal cartilage or disc cartilage.
  • endogenous cells are used with and without carrier material and on the other hand only carrier materials introduced into the defect, depending on the indication resorbable or non-absorbable materials can be used.
  • cartilage and bone cell transplantation which is used to treat cartilage and bone defects.
  • the potential of the cartilage and bone cells is used to build new tissue in vivo .
  • cartilage or bone biopsies are removed from the patient, cartilage or bone cells are isolated therefrom, multiplied by cell cultivation, and then the patient is transplanted the cells in the region of the tissue defect, for example by injection. There they form new tissue and thus fill up the defect completely.
  • tissue is built up in the body after application of the cell transplants or introduction of the carrier materials.
  • tissue engineering is to prefabricate the body's own tissue in vitro .
  • a large number of methods are known from the literature (cf. DE 195 40 487 . WO 97/46665 . DE 197 52 900 . US 5,932,459 ), which either require special devices or carriers, involve many process steps or require the addition of growth-promoting compounds which are foreign substances to the body.
  • the object of the present invention was therefore to provide the simplest possible method for the production of typical cartilage or bone tissue, with which vital, three-dimensional and mechanically stable tissue can be produced, which is suitable for transplantation and a rapid growth in the body or a fast Filling the cartilage or bone defect ensured.
  • the tissue produced in vitro should as far as possible trigger no immunological reactions in the organism receiving the transplant.
  • US 5,723,331 discloses cylindrical cell aggregates formed in differently preformed wells as needed.
  • the preformed wells consist of flat floors.
  • flat cartilage grafts are formed, which can be integrated macrosurgically into the wound base.
  • US 5,723,331 is cultured exclusively with xenogenic sera.
  • New York describes the formation of cartilage clusters after 4 days of culture and that chondrocytes multiply in culture within the first 15 days.
  • tissue biopsies or samples or mesenchymal stem cells are used as starting material.
  • the tissue-building cells are isolated by enzymatic digestion of the tissue, by emigration, or by reagents that recognize target cells by conventional methods.
  • These cells are then cultured according to the invention in a simple manner with conventional culture medium in cell culture vessels with hydrophobic surface and tapered soil in suspension stationary until a three-dimensional cell aggregate is formed, which is at least 40% by volume, preferably at least 60% by volume to 95% by volume.
  • extracellular matrix (ECM) in which differentiated cells are embedded.
  • the resulting cell aggregate has an outer area in which cells capable of proliferation and migration are present.
  • FIG. 1 and la This structure of the cell aggregates obtained according to the invention is illustrated by the microscopic images in FIG Fig. 1 and la, where Fig. 1 represents the sectional enlargement of the cross-section of a cell aggregate according to the invention with vP as a zone of the occurrence of the first tissue-specific matrix proteins and M as a zone of formation of tissue-specific matrix proteins, and Fig. 1a shows the entire cell aggregate with the outer zone of proliferative and migratory cells P (zone of expression of protein S 100).
  • the cells inside the aggregates differentiate and spheroids are formed, consisting of ECM, differentiated cells, and a peripheral proliferation zone.
  • the process of forming this tissue-specific matrix with embedded cells is very similar to the process of tissue formation and remodeling in the body.
  • tissue histology is created that is very similar to natural tissue.
  • the supply of cells inside the spheroids, as in natural cartilage also, only by the diffusion of nutrients.
  • the zone of proliferative and migratory cells forms at the edge of the spheroids.
  • This zone has the inestimable advantage that after introducing the spheroids into defects, the cells located in this marginal zone are able to migrate and actively make contact with the surrounding tissue or allow integration of the tissue formed in vitro into its environment.
  • the tissue-specific cell aggregates produced are outstandingly suitable for the treatment of tissue defects and for the rebuilding of tissue in vitro and in vivo .
  • tissue defect to be treated it may be advantageous to transplant even larger tissue pieces in order to achieve a faster filling of the defect.
  • at least two, but more preferably more, of the cell aggregates obtained are fused by cultivating them together to the desired size under the same conditions and in the same culture vessels as described above.
  • Fig. 1b shows two spheroids to be fused after one day.
  • Fig. 1c shows that a few hours later, the boundary between the two spheroids is no longer recognizable. After another week, the spheroids are completely fused and a larger piece of in vitro tissue has been formed ( Fig. 1d ).
  • the structure of the larger cell aggregates thus obtained is identical to that of the initially obtained spheroids. They can contain up to a maximum of 95% ECM and all cells contained in the obtained tissue are vital.
  • the resulting cartilage or bone tissue is extremely stable.
  • the cell aggregates can be compressed to 3/4 of its diameter, without breaking or, for example, when injected into the body falling apart by means of a cannula. It is possible to remove these pieces of tissue from the cell culture vessel with tweezers or a pipette.
  • the cells obtained by the patient are first multiplied in monolayer culture in a manner known per se.
  • the passage of the cells in monolayer culture is kept as low as possible.
  • the monolayer grown cells are harvested and cultured according to the method of the invention in suspension, as described above.
  • the cell culture medium it is possible to use conventional medium, for example Dulbecco's MEM, with the addition of serum, both for the suspension culture and for the monolayer culture.
  • DMEM and Hams are used in the ratio 1: 1.
  • the autologous serum of the patient is preferably used as the serum. It is also possible to use xenogeneic or allogeneic serum.
  • no antibiotics, fungistatics or other auxiliaries are added to the culture medium. It has been shown that only the autogenous, xenogeneic or allogeneic cultivation of the cells and cell aggregates as well as the cultivation without antibiotics and fungistatics allows an unaffected morphology and differentiation of the cells in the monolayer culture and an undisturbed formation of the specific matrix in the cell aggregates. Furthermore, all the immunological reactions are excluded by the omission of all additives during manufacture after introduction of the tissue produced in vitro into the human and also animal organism.
  • three-dimensional cell aggregates are already present after two days of suspension cultivation according to the invention obtained tissue-specific properties.
  • the size depends on the number of cells introduced per volume of culture medium. If, for example, 1 ⁇ 10 7 cells are introduced into 300 ⁇ l of culture medium, three-dimensional spheroids of about 500-700 ⁇ m diameter are formed within 1 week. For a 1 cm 2 tissue defect, approximately 100 such spheroids would have to be transplanted, eg injected.
  • the other possibility is the in vitro fusion of the small cell aggregates to larger - as described above - and the introduction of these into the defect.
  • between 1 ⁇ 10 4 and 1 ⁇ 10 7 cells are used in 300 ⁇ l culture medium for the preparation of the small cell aggregates, particularly preferably 1 ⁇ 10 5 cells.
  • the spheroids formed after a few days are then cultured for at least 2-4 weeks depending on the cell type and the patient-specific characteristics in the appropriate culture medium to induce the formation of the tissue-specific matrix.
  • individual spheroids can then be fused from about one week of cultivation to increase the size of the tissue piece.
  • cell culture vessels for the culture according to the invention in suspension, those having a hydrophobic, ie adhesion-preventing, surface, such as e.g. Polystyrene or Teflon, are used.
  • Non-hydrophobic surface cell culture vessels may be rendered hydrophobic by coating with agar or agarose. Further additives are not required.
  • cell plates serve as cell culture vessels.
  • 96-well plates can be used for the production of the small cell aggregates and 24-well plates can be used for the production of the fused aggregates.
  • the cell culture vessels must have a tapered, preferably curved bottom. It has been shown that the tissue according to the invention does not form in vessels with a flat bottom. Obviously, the well serves to find the cells.
  • the present invention also discloses the cartilage or bone tissue prepared by the above-described method which can be used as an autogenous, xenogeneic or allogenic transplant material in the treatment of cartilage or bone defects and degenerative diseases such as osteoarthritis or rheumatism.
  • the cell aggregates according to the invention produced in vitro are injected by injection into the diseased or degraded tissue. This requires the injection needle or a other suitable application system have at least the diameter of the spheroids.
  • the cells in the zone of proliferative and migratory cells at the periphery of the spheroids grow rapidly into the surrounding tissue and allow rapid integration of the tissue produced in vitro and represent a potential for the regeneration of tissue in the vicinity of the spheroids, since these cells are still proliferating and matrix form.
  • This treatment can be done arthroscopically in advance of previous procedures in tissue engineering.
  • the present invention also discloses therapeutic compositions comprising the cartilage or bone tissue of the invention, e.g. B. injection solutions.
  • the present invention also discloses the use of the cartilage or bone tissue of the invention for testing various factors, e.g. Active ingredients and physical factors, e.g. influence the formation and differentiation of matrix and cells, the physical factors being e.g. Pressure or electrical fields.
  • the cell spheroids are prepared according to the invention and in different stages of maturity the drugs to be tested are added and characterized various parameters of Sphotroidentstehung and maturation. These tests are very patient specific and allow for individual diagnosis compared to conventional drug testing on animals or tumor systems using only autologous material.
  • the patient is biopsied from one area of hyaline, healthy cartilage. From this biopsy, the chondrocytes are isolated by enzymatic digestion by incubation with collagenase solution. After separation of the isolated cells from the undigested cartilage tissue, they are transferred to cell culture flasks and added with the addition of DMEM / Ham's F12 culture medium (1/1) and 10% autologous serum of the patient 37 ° C and 5% CO 2 incubated. Twice weekly, a medium change is performed. After reaching the confluent stage, the cell layer is washed with physiological saline and harvested by trypsin from the cell culture surface. After another wash, 1 ⁇ 10 5 cells are transferred into each cell culture vessel which is coated with agarose.
  • the first cells After one day, the first cells have been arranged in aggregates. These aggregates are supplied with fresh medium every 2 days and cultured for at least 2 weeks. Already after one week collagen type II and proteoglycans were detected in the aggregates. For this a specific antibody against collagen type II was used. The primary antibody bound to type II collagen was detected using a second antibody and coupled ABC system. That is, the second antibody is coupled via avidin-biotin, the enzyme alkaline phosphatase, which converts the substrate fuchsin, wherein a red dye is formed.
  • proteoglycans were detected by gold staining.
  • Collagen type II and proteoglycans are components of the cartilage matrix in vivo and represent the most important structural proteins that are crucial for the function of cartilage.
  • the protein S 100 specific for cartilage cells was detected in the outer layer of the aggregates. S 100 is not expressed in bone tissue and connective tissue. Only these tissues could arise here. Thus, it was clearly demonstrated that the developed tissue is cartilage tissue.
  • the tissue prepared in Example 1 (about 200 spheroids of each 1 * 10 5 cells) was taken up in physiological saline and in a ca. 1 cm 2 large, with periosteum Covered cartilage defect of the subject injected. It has been found that the cartilage defect is already filled with cartilage within 1 to 2 months, whereas the previous transplantation of young, only in vitro propagated cartilage cells, the repletion of a defect of such size only after 6-12 months.
  • the in vitro produced cartilage tissue according to the invention ensures the rapid integration of the tissue pieces produced by the proliferation-capable and migratory cells in the outer layer of the aggregates.
  • the structure and function of the tissue pieces also allows the rapid repair of defects and degenerated cartilage tissue.
  • a bone biopsy is taken from the area of cancellous bone. From this biopsy, the osteoblasts are isolated by enzymatic digestion by incubation with collagenase solution. After separation of the isolated cells from the undigested bone tissue, they are transferred to cell culture flasks and incubated at 37 ° C and 5% CO 2 with the addition of DMEM / Ham's F12 culture medium (1/1) and 10% autologous serum of the patient. Twice weekly, a medium change is made. After reaching the confluent stage, the cell layer is washed with physiological saline and harvested by trypsin from the cell culture surface. After another wash, 1 ⁇ 10 5 cells are transferred into each cell culture vessel which is coated with agarose. After one day, the first cells have been arranged in aggregates. These aggregates are supplied with fresh medium every 2 days and cultured for at least 2 weeks.
  • collagen type I and proteoglycans were detected in the aggregates.
  • a specific antibody against collagen type I was used.
  • the detection of collagen I has unequivocally proven that it is not cartilaginous tissue.
  • the primary antibody bound to Type I collagen was detected using a second antibody and coupled ABC system. That is, the second antibody is coupled via avidin-biotin, the enzyme alkaline phosphatase, which converts the substrate fuchsin, wherein a red dye is formed.
  • proteoglycans were detected by gold staining as in Example 1.
  • Collagen type I and proteoglycans are components of the bone matrix in vivo and represent the most important structural proteins that are crucial for the function of the bone.
  • the tissues prepared in Example 3 (about 50 pieces) are taken up in physiological saline and injected into the area of a hard-healing bone fracture. It was found that the process of bone healing could be induced and new bone tissue was formed after only 3 weeks, whereas failure to treat the defect would result in fracture healing only after several months.
  • the bone tissue grown in vitro thus ensures the rapid integration of the tissue into the surrounding tissue and the filling of bone defects.
  • the structure and function of the tissue pieces allows the healing of bone defects, the induction of osteosynthesis and the treatment of degenerative bone disease.

Claims (6)

  1. Procédé de production in vitro d'un agrégat cellulaire de tissu vital tridimensionnel cartilagineux ou osseux, qui est sphéroïde, caractérisé en ce que lesdites cellules cartilagineuses, osseuses ou cellules souches mésenchymateuses provenant d'organismes humains ou animaux sont cultivées de façon stationnaire en suspension dans des récipients de culture cellulaire ayant une surface hydrophobe et une base effilée, avec addition de sérum autogène ou de sérum autologue, sans facteurs de croissance, antibiotiques ou fongistatiques, jusqu'à obtention d'un agrégat cellulaire sphéroïdal contenant au moins 40 % du volume de la matrice extracellulaire (MEC) dans laquelle les cellules différenciées sont intégrées, et présentant une zone externe sur laquelle se trouvent des cellules capables de prolifération et de migration.
  2. Procédé selon la revendication 1, caractérisé en ce que les cellules cartilagineuses ou osseuses provenant des explants sont d'abord multipliées en culture monocouche avant d'être cultivées en suspension selon la revendication 1.
  3. Procédé selon la revendication 1 ou 2, caractérisé en ce qu'en fonction de la taille de tissu désirée, deux ou plusieurs des agrégats cellulaires sphéroïdaux obtenus sont fusionnés par culture associée supplémentaire en suspension dans des récipients de culture cellulaire ayant une surface hydrophobe et une base effilée.
  4. Procédé selon une quelconque des revendications 1 à 3, caractérisé en ce que des plaques à puits servent de récipients de culture cellulaire.
  5. Procédé selon une quelconque des revendications 1 à 4, caractérisé en ce que les récipients de culture cellulaire ayant une surface non hydrophobe sont rendus hydrophobes par revêtement avec l'agar-agar ou l'agarose.
  6. Procédé selon une quelconque des revendications 1 à 5, caractérisé en ce que la culture en suspension est effectuée jusqu'à obtention d'un agrégat cellulaire contenant au moins 60 % de MEC.
EP01913858.5A 2000-03-13 2001-03-09 Procede permettant de produire i in vitro /i du tissu cartilagineux ou osseux tridimensionnel vivant Expired - Lifetime EP1265986B1 (fr)

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Application Number Priority Date Filing Date Title
CY20161100246T CY1117464T1 (el) 2000-03-13 2016-03-23 Μεθοδος για την in vitro παραγωγη τρισδιαστατου ζωτικου χονδρινου ή οστικου ιστου

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10013223A DE10013223C2 (de) 2000-03-13 2000-03-13 Verfahren zur in vitro-Herstellung von dreidimensionalem, vitalem Knorpel- oder Knochengewebe und dessen Verwendung als Transplantationsmaterial
DE10013223 2000-03-13
PCT/EP2001/002698 WO2001068811A2 (fr) 2000-03-13 2001-03-09 Procede permettant de produire in vitro du tissu cartilagineux ou osseux tridimensionnel vivant, et son utilisation comme matiere de greffe

Publications (2)

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EP1265986A2 EP1265986A2 (fr) 2002-12-18
EP1265986B1 true EP1265986B1 (fr) 2016-02-24

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US (1) US7887843B2 (fr)
EP (1) EP1265986B1 (fr)
AU (1) AU2001239287A1 (fr)
CA (1) CA2403120A1 (fr)
CY (1) CY1117464T1 (fr)
DE (1) DE10013223C2 (fr)
DK (1) DK1265986T3 (fr)
ES (1) ES2566053T3 (fr)
WO (1) WO2001068811A2 (fr)

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US20030153078A1 (en) 2003-08-14
DE10013223C2 (de) 2002-07-18
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WO2001068811A3 (fr) 2001-12-27
ES2566053T3 (es) 2016-04-08
US7887843B2 (en) 2011-02-15
CY1117464T1 (el) 2017-04-26
CA2403120A1 (fr) 2002-09-13
DE10013223A1 (de) 2001-10-11
EP1265986A2 (fr) 2002-12-18
DK1265986T3 (en) 2016-04-04

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